The program project represents a comprehensive collaborative effort the ultimate goal of which is to develop novel drug analogs which produce their therapeutic effects by acting on the cannabinoid receptors. A central hypothesis of this program is that the recent availability of such receptor(s) offers the opportunity to rationally design analogs with a high degree of selectivity for inducing certain actions of cannabinoids including analgesia, inhibition of vomiting and reduction of intraocular pressure and immuno-modulation without their undesirable psychoactive effects. Similarly, there will be an opportunity for developing novel ligands which can successfully block the actions of cannabinoids. Such a process will require detailed knowledge of the molecular, biochemical and anatomical features of this receptor and its subtypes which are associated with cannabinoid activity. The receptor active sites could thus be used as template(s) for the successful design of these novel analogs. Obtaining intimate knowledge of the receptors' structure and function will require an interdisciplinary approach which will be accomplished through concerted collaborative efforts between several laboratories with a commitment for cannabinoid research and/or the high degree of technical expertise required for an effective approach to this problem. Strong collaborative interactions will be emphasized with the following major specific aims (1) the development of high affinity ligands; for the receptor(s) which will be used for obtaining molecular information on the cannabinoid site(s) of action. The group of ligands to be developed will encompass all four classes of molecules which are associated with cannabimimetic activity including classical cannabinoids; (CCs), non-classical cannabinoids (NCCs), aminoalkylindoles (AAls) and arachidonic acid amides (AAAs). (2) The expression isolation, purification and reconstitution of the cannabinoid receptor(s) and its mutants in viruses and bacteria. (3) Obtaining receptor active sites(s) with the help of high affinity covalent receptor ligands and by determining the amino acid residues with which the ligands reacted (using photoaffinity labeling). Determination of the specific sites on the receptor (length = 472 amino acids) that are photoaffinity labeled will be accomplished using high performance liquid chromatographyand mass spectrometry. Several different mass spectrometric techniques are being used. MALDI-TOF mass spectrometric mapping of proteolytic digests of the receptor prior to and after reaction. This measurement will provide definition of the binding site to within a particular proteolytic peptide. 11 LC-ESI mass spectrometry of proteolytic digests of the receptor. This measurement will provide similar information to that obtained in I above. However, since we will split the flow, fractions of interest will be collected for further study. 111. MALDI-ITMS and ESI-triple quadrupole tandem mass spectrometry of peptides that have been identified to be modified by the ligands of interest (either through observed mass shifts in I & 11 above or by radioactive labeling). In this context, we have recently defined the binding site of E. coli RecA to single stranded DNA during the process of homologous recombination. In this case, photocrosslinking was used and the binding site determined by MALDI-ITMS/MS (at the single amino acid level) was independently confirmed by Edman sequencing.